Design of potent inhibitors of human β-secretase. Part 1

Article abstract of DOI:10.1016/j.bmcl.2006.09.092

We describe a novel series of potent inhibitors of human β-secretase. These compounds possess the hydroxyethyl amine transition state isostere. A 2.5 A crystal structure of inhibitor 32 bound to BACE is provided.

Full text of DOI:10.1016/j.bmcl.2006.09.092

Bioorganic & Medicinal Chemistry Letters 17 (2007) 73–77
Design of potent inhibitors of human b-secretase. Part 1
John N. Freskos,a,* Yvette M. Fobian,a,à Timothy E. Benson,a,à Michael J. Bienkowski,a
David L. Brown,a Thomas L. Emmons,a Robert Heintz,§ Alice Laborde,§
Joseph J. McDonald,a Brent V. Mischke,a John M. Molyneaux,a Joseph B. Moon,a,à
Patrick B. Mullins,a D. Bryan Prince,– Donna J. Paddock,b Alfredo G. Tomassellia and
Gregory Winterrowd
aPfizer Inc., 700N. Chesterfield Pkwy., St. Louis, MO 63198, USA
bPfizerInc., 2800 Plymouth Rd, Ann Arbor, MI 48105, USA
Received 11 July 2006; revised 23 September 2006; accepted 29 September 2006
Available online 4 October 2006
Abstract—We describe a novel series of potent inhibitors of human b-secretase. These compounds possess the hydroxyethyl amine
˚
transition state isostere. A 2.5 A crystal structure of inhibitor 32 bound to BACE is provided.
ꢀ 2006 Elsevier Ltd. All rights reserved.
b-Secretase or b-site APP-cleaving enzyme (BACE) has
been identified as an aspartyl protease responsible for
the first mandatory proteolytic step to generate amyloi-
dogenic b-peptides from the naturally occurring amyloid
precursor protein (APP).1–4 Site specific cleavage of APP
(both wild-type and Swedish mutation) by b-secretase
yields the 99 amino acid C-terminal fragment which is
further processed by the c-secretase/presenilin(s) com-
plex at residue 711 or 713 to liberate a peptide of 40 res-
idues (Ab40) or of 42 residues (Ab42) Generation of Ab is
thought to be causal for the pathology and cognitive de-
cline in Alzheimer’s disease AD.5,6
Figure 1. Evolution of the isophthalate hydroxyethyl amine series.
Our goal was to discover and develop potent, safe, and
efficacious agents to block formation of Ab plaque via
Interestingly in this series phenyl (Phe) at P1 is more po-
tent than isobutyl (Leu), which is found at P1 in the
the b-secretase pathway and thus block the onset and/
or progression of AD. Recently, we disclosed a potent
Swedish mutant that helped in the initial identification
series of BACE inhibitors utilizing a hydroxyethyl amine
(HEA) transition state isostere (TSIs)7a–7d see Figure 1.
of BACE, and 3,5-difluorophenyl was found to be opti-
mum. It typically gives ꢀ10 to 100 · boost in potency
over phenyl while the m-iodo for m-methoxy is ꢀ2 ·
(1 vs 2). It is also noteworthy how important it is to
occupy S-3 in order to maintain potency (3 vs 4). After
Keywords: b-Secretase; Hydroxyethylamine (HEA) isostere.
Corresponding author. Tel.: +1 636 247 7406; fax: +1 636 247
5401; e-mail: john.n.freskos@pfizer.com
the initial SAR of the isophthalide series was described
*
we found that others had initiated similar work on the
HEA series of inhibitors.8 A recent review on synthesis
of BACE inhibitors has also appeared.9
Deceased.
Co-authors.
à
§
Retired.
Present address: Department of Chemistry, University of Oklahoma,
Norman, OK 73019, USA.
–
In the present letter, we describe our attempts to replace
the potent isophthalate N-terminus with novel acyclic
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.092

74
J. N. Freskos et al. / Bioorg. Med. Chem. Lett. 17 (2007) 73–77
scaffolds that maintain good enzyme and cellular poten-
cy for BACE and selectivity for other aspartyl proteases
in particular the ubiquitous protease Cathepsin-D (Cat-
D).7d Also we will outline the initial SAR exploration,
which was done in the phenylalanine series at P1, while
a following paper will discuss utilization of the more po-
tent 3,5-difluorophenylalanine at P1 to increase potency
for BACE as well as optimization of the S2 and S3 side
chains to improve selectivity against Cat-D while main-
taining potency for BACE.
(Scheme 2). Oxidation with OXONEꢁ yielded the
desired sulfone acid 10 which was then coupled to the
C-terminal fragment 7 using standard peptide coupling
(EDC/HOBT) conditions. Removal of the Boc group
with 4 N HCl-dioxane afforded the targeted two-carbon
sulfone HEA inhibitor 11 (n = 1). The three-carbon
spacer sulfone was prepared in a similar fashion starting
with an SN20 ring opening of butyrolactone (9) with the
appropriate thiol. Oxidation to the sulfone followed by
coupling and deprotection afforded the targeted three
carbon sulfone inhibitor (n = 2).
We initially decided to examine acyclic compounds with
either a two or three atom spacer between the N-termi-
nal carbonyls. From crystallographic data obtained on 3
we knew that the carbonyl of the dipropyl amide was a
key H-bond acceptor with the side-chain hydroxy of
Thr-232.7b,c Assuming it was critical to maintain this
interaction we thus decided to explore acyclic sulfones
and sulfonamides with varying spacer atoms between
the amide carbonyl and the SO2 group.
The fully assembled acyclic sulfonamide inhibitors were
prepared by reacting potassium thioacetate with either
ethyl 3-bromopropionate or ethyl 4-bromobutyrate
(12) to give the corresponding thioacetate ester (Scheme
3). Chlorine oxidation to the sulfonyl chloride followed
by coupling with the appropriate amine and base hydro-
lysis of the ester gave the desired N-terminal fragment 2
or 3 carbon spacer sulfonamide acid 13. Coupling to the
C-terminal fragment 7 using standard peptide coupling
(EDC/HOBT) conditions and removal of the Boc group
with 4 N HCl in Dioxane afforded the targeted sulfon-
amide HEA inhibitor 14.
The targeted sulfone and sulfonamides were prepared
using a common intermediate 7 (Scheme 1). The reac-
tion of epoxide 510 with excess 3-methoxybenzylamine
in refluxing 2-propanol afforded the secondary amine 6
in high yield. Protection with (Boc)2O followed by
removal of the Cbz group by catalytic hydrogenation
afforded the desired C-terminal coupling fragment 7.
Table 1 summarizes the enzyme inhibition results using
a fluorescence polarization assay (FP)11 to determine
the IC50 values for inhibition of recombinant human
BACE and Cat-D. In contrast to the previous SAR
demonstrating that a three-carbon spacer was more
potent than two- or four-,12 both the sulfone and sulfon-
amide analogues were more potent with a two-carbon
spacer. Evidently, the difference in the bond length for
The synthesis of the fully assembled acyclic sulfone
inhibitors was straightforward. For the two-carbon
spacer the appropriate thiol was reacted with b-chloro
propionic acid (8) to afford the desired sulfide acid
Scheme 1.
Scheme 2.
Scheme 3.

J. N. Freskos et al. / Bioorg. Med. Chem. Lett. 17 (2007) 73–77
Table 1. Enzyme inhibition values for acyclic sulfones and sulfonamides
75
Compound
n
R1
BACE-FP IC50 (nM)
CatD-FP IC50 (nM)
Sulfones
15
16
3
2
1
2
1
1
2
1
1
Phe
Phe
>20,000
4131
5214
11,202
>20,000
6042
17
18
Phe
Cyclohexyl
Cyclohexyl
Methyl
Isopentyl
Isopentyl
4-Heptyl
>20,000
3670
>20,000
6946
7593
19
20
>20,000
>20,000
5614
21
22
>20,000
10,986
9669
23
596
Sufonamides
24
25
26
27
28
29
2
1
1
1
2
1
Piperidinyl
Piperidinyl
6461
3785
5015
1090
7950
484
>20,000
>20,000
2116
Isopentylamino
Dipropylamino
Dipropylamino
Dibutylamino
4900
11,700
4880
a CAS bond versus a CAC bond required one less car-
bon spacer to appropriately position one of the oxygens
in the sulfonyl group for the desired interaction with
Thr-232. Within the more potent two carbon series,
potency against BACE increased for analogues with al-
kyl substitution at the N-terminus over either aryl or
cyclic substituted analogues. Furthermore, potency for
BACE increased when the substitution more closely
mimicked the diisopropylamide N-terminus of the iso-
phthalate series.
Within both series it was possible to achieve equipotent
BACE inhibitors by replacing the isophthalate N-termi-
nus of 1 with an appropriate acyclic sulfone (23) or sul-
fonamide (29). Additionally each of these analogues
maintained excellent potency in the HEK-293 Swe amy-
loid inhibition cell assay13 with 314 and 130 nM values,
respectively.
Figure 2.
the racemic Cbz-analogue 32 versus the sulfone ana-
logue 23 with no P2 substituent (Fig. 3).
In an effort to further improve potency for BACE by
accessing the S2 pocket as shown for the isophthalate
HEA inhibitor 3, the a-Cbz-amine 32 was proposed
(Fig. 2). As shown in Scheme 4, the Michael acceptor
methyl-2-acetamidoacrylate (30) was reacted with
4-mercaptoheptane and then oxidized with OXONEꢁ
to give the b-sulfone amino acid. Hydrolysis of the
methyl ester and N-acyl group with concentrated hydro-
chloric acid in glacial acetic acid afforded the free amino
acid which was protected using Cbz-succinimide to yield
31. Coupling of 31 with the C-terminal fragment 7 fol-
lowed by removal of the Boc group then afforded the de-
sired inhibitor, 32. When tested in the fluorescence
polarization assay, 32 inhibited BACE with an
IC50 = 112 nM. This compound was even more potent
for Cat-D with an IC50 = 67 nM. This represented
approximately a 5-fold increase in BACE activity for
Modeling of 32 in the active site of BACE predicted the
preferred stereochemistry for substitution at the a posi-
˚
tion to be S. A subsequent 2.5 A X-ray crystal structure
of recombinant human BACE expressed and purified
from Escherichia coli14 and co-crystallized in the pres-
ence 32 (a racemic mixture) resulted in binding of a sin-
gle isomer confirming the preferred stereochemistry was
indeed S. One of the sulfone oxygens provides a hydro-
˚
gen bond acceptor for the Thr232 NH (3.0 A). The Cbz
group projects toward P2 but reaches past Arg235 into
the solvent. The carbonyl of the Cbz is involved in a
long water mediated interaction to the main chain NH
˚
of Asn233 (3.7 A), and the benzyl moiety is nestled adja-
cent to Gln73. The absence of direct interactions with
Arg235 most likely accounts for the lack of specificity
with respect to Cat-D. Other interactions between 32

76
J. N. Freskos et al. / Bioorg. Med. Chem. Lett. 17 (2007) 73–77
Scheme 4.
References and notes
1. Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.;
Tory, M. C.; Pauley, A. M.; Brashier, J. R.; Stratman, N.
C.; Mathews, W. R.; Buhl, A. E.; Carter, D. B.;
Tomasselli, A. G.; Parodi, L. A.; Heinrikson, R. L.;
Gurney, M. Nature 1999, 402, 533.
2. Sinha, S.; Anderson, J. P.; Barbour, R.; Basi, G. S.;
Caccavello, R.; Davis, D.; Doan, M.; Dovey, H. F.;
Frigon, N.; Hong, J.; Jacobson-Croak, K.; Jewett, N.;
Keim, P.; Knops, J.; Lieberburg, I.; Power, M.; Tan, H.;
Tatsuno, G.; Tung, J.; Schenk, D.; Seubert, P.; Suomen-
sari, S. M.; Wang, S.; Walker, D.; Zhao, J.; McConlogue,
L.; John, V. Nature 1999, 402, 537.
3. Vasser, R.; Bennett, B. D.; Babu-Khan, S.; Mendiaz, E.
A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.;
Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.;
Lile, J.; Jarosinski, M. A.; Biere, A. L.; Curran, E.;
Burgess, T.; Louis, J. C.; Collins, F.; Treanor, J.; Rogers,
G.; Citron, M. Science 1999, 286, 735.
Figure 3. Compound 32 bound to human recombinant BACE.
4. Lin, X.; Koelsch, G.; Wu, S.; Downs, D.; Dashti, A.;
Tang, J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1456.
5. Selkoe, D. J. Nature 1999, 399(6738 Suppl), A23.
6. Sinha, S.; Lieberburg, I. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 11049; Citron, M. Science 1999, 286, 735.
7. (a) Maillard, M.; Hom, R.; Gailunas, A.; Jagodzinska, B.;
Fang, L. Y.; John, V.; Freskos, J. N.; Pulley, S. R.; Beck,
J. P.; Tenbrink, R. E. 2002, WO Patent 0202512.; (b)
Hom, R. K; Maillard, M; Mamo, S.; Dappen, M. A.;
Brogley, L.; John, V.; Thorsett, E. D.; Benson, T. E.;
Abstracts of Papers, 232nd National Meeting of the
American Chemical Society, San Francisco, CA. Septem-
ber 10–14, 2006.; (c) Maillard, M; Hom, R. K.; Benson, T.
E,; Moon, J. B.; Mamo, S.; Bienkowski, M.; Tomasselli,
A. G.; Woods, D. D.; Prince, D. B.; Paddock, D. J.;
Emmons, T. L.; Galiunas, A.; Tucker, J.; Dappen, M. A.;
Brogley, L.; Thorsett, G.; Jewett, N.; Sinha, S. John, V.,
J. Med. Chem., in preparation.; (d) Freskos, J. N.;
Aquino, J.; Brown, D. L.; Fang, L.; Fobian, Y. M.;
Gailunas, A. F.; Gunn, A.; John, V.; Romereo, A. G.;
Tucker, J.; Tung, J.; Walker, D. 2002 WO Patent
020988849.
are similar to those previously observed for other
BACE-HEA co-crystal structures.7b,c,15
In summary, we have described a series of potent BACE
inhibitors utilizing several novel acyclic scaffolds to re-
place the isophthalate N-termini of existing hydroxyeth-
ylamine inhibitors. Both access the critical S3 subsite of
BACE. Equipotent inhibition for BACE was achieved
through the proper choice of carbon linker and appro-
priate N-termini substitution. Additional potency was
achieved by occupying the S2 pocket of the enzyme
through incorporation of an a-side chain on the N-ter-
minus of the acyclic scaffold. Finally, a preferred stereo-
chemistry for this substitution was predicted by
modeling and subsequently confirmed by X-ray
crystallography.
8. Stachel, Shawn J.; Coburn, Craig A.; Steele, Thomas G.;
Crouthamel, Min-Chi; Pietrak, Beth L.; Lai, Ming-Tain;
Holloway, M. Katharine; Munshi, Sanjeev K.; Graham,
Samuel L.; Vacca, Joseph P. Bioorg. Med. Chem. Lett.
2006, 16, 641; Stachel, Shawn J.; Coburn, Craig A.; Steele,
Thomas G.; Jones, Kristen G.; Loutzenhiser, Elizabeth F.;
Gregro, Alison R.; Rajapakse, Hemaka A.; Lai, Ming-
Tain; Crouthamel, Ming-Chih; Xu, Min; Tugusheva,
Katherine; Lineberger, Janet E.; Pietrak, Beth L.; Espes-
eth, Amy S.; Shi, Xiao-Ping; Chen-Dodson, Elizabeth;
Holloway, M. Katharine; Munshi, Sanjeev; Simon, Adam
J.; Kuo, Lawrence; Vacca, Joseph P. J. Med. Chem. 2004,
47, 6447.
Acknowledgments
The authors would like to acknowledge Dr. Daniel Rich
for his advice and support of this work. Dr. T.E.B.
would like to acknowledge the use of the IMCA-CAT
beamline 17-ID at the Advanced Photon Source which
is supported by the companies of the Industrial Macro-
molecular Crystallography Association through a con-
tract with the Center for Advanced Radiation Sources
at the University of Chicago. Use of the Advanced Pho-
ton Source was supported by the U.S. Department of
Energy, Office of Science, Office of Basic Energy Scienc-
es, under Contract No. W-31-109-Eng-38.
9. Ghosh, A. K.; Kumaragubaran, N.; Tang, J. Curr. Topics
Med. Chem. 2005, 5, 1609; Baxter, E. W.; Reitz, A. B.
Annu. Reports Med. Chem. 2005, 1, 35.

J. N. Freskos et al. / Bioorg. Med. Chem. Lett. 17 (2007) 73–77
77
10. Getman, D. P.; DeCrescenzo, G. A.; Heintz, R. M.; Reed,
12. Unpublished results of Dr. John Tucker.
13. Dovey, H. F. et al. J. Neurochem. 2001, 76, 173.
14. Tomasselli, A. G.; Benson, T. E. et al., in preparation.
15. Coordinates for the complex of BACE with inhibitor 32
have been deposited in the Protein data bank
(www.rcsb.org) under PDB ID 2HIZ.
K. L.; Talley, J. J.; Bryant, M. L.; Clare, M.; Houseman,
K. A.; Marr, J. J.; Mueller, R. A.; Vazquez, M. L.; Shieh,
H-S.; Stallings, W. C.; Stegeman, R. A. J. Med. Chem.
1993, 36, 288.
11. Unpublished work of Dr. Mike Bienkowski and Dr. Alice
Laborde.